<p>Nascent proteins begin to fold during their synthesis, while still attached to the ribosome. The dynamic nature of ribosome–nascent chain complexes (RNCs) poses a challenge for conventional structural biology approaches, limiting our understanding of dynamic cotranslational events. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) is a powerful label-free technique for studying the conformational equilibria and refolding of full-length proteins with peptide resolution. However, the large size of the ribosome and the need for stable, highly homogeneous samples have hindered the application of HDX-MS to RNCs. Here we present a strategy for analysing conformational dynamics and interactors of <i>Escherichia coli</i> RNCs using HDX-MS. High-quality RNCs are obtained through the gentle lysis of high-density cultures expressing uniformly stalled ribosomes, followed by ultracentrifugation and tag-based affinity purification. Peptide-resolution information on protein conformational dynamics is obtained by pulse deuterium labeling, quenching with an RNA-compatible low pH buffer and offline digestion with pepsin. Extensive data analysis with use of specific internal controls allows for the confident assignment of mass spectra to specific peptides, ensuring good coverage of the nascent chain and ribosomal proteins. This method provides a valuable complement to existing structural techniques such as cryo-electron microscopy and nuclear magnetic resonance, and enables detailed characterization of large, partially structured nascent chains and their interactions with the ribosomal proteins and molecular chaperones. The protocol takes 1–3 months, from sample preparation and data acquisition to data analysis, and requires standard expertise in cloning and protein purification and intermediate expertise in HDX-MS.</p>